The laboratory mouse is a popular model for the study of human disease. In the recent past, the mouse has also become widely used for the analysis of mammalian gene function. The genome of the mouse is relatively accessible and, thus, many transgenic mice have been engineered. Homologous recombination in mouse embryonic stem cells has been a convenient vehicle for the creation of these transgenic mice. One major, rate-limiting factor for the determination of gene function has been the lack of access to validated, germline competent mouse ES cell lines from various genetic backgrounds. The newly launched NIH-sponsored program entitled "Knockout Mouse Project (KOMP)" is striving to generate a complete or nearly complete set of knockout mutations of the genes in the mouse genome on the C57Bl/6 genetic background. Our proposal is complementary to the KOMP project in that one of the lines we are proposing to use is a C57Bl/6 (line LB10) and the other line is an F1 hybrid C57Bl/6 x 129 (line LC3). Both lines are homozygous for a ubiquitously expressed GFP transgene. The ability to compare results across laboratories requires that studies be performed on the same starting material. Ample evidence for genotypic and phenotypic changes, partially induced through improper handling during culture, exists regarding the propagation of ES cell lines. Here, we propose to standardize, propagate, and thoroughly characterize two mouse ES cell lines, LC3 and LB10. The ultimate goal of this application is to make ES cell lines from useful mouse models readily accessible to the scientific community. Specifically, our aims are: Aim I. Establish Master and Working Cell Banks for each mouse ES cell line. Aim II. Fully characterize the two mouse ES cell lines and prepare them for distribution. Work under Aim I will include standardization of culture conditions for the two lines and expansion of the cells under those conditions, while under Aim II we will monitor for changes in pluripotency during in vitro propagation. Our ultimate test for pluripotency will be assaying the ES cell lines for their ability to populate the germ lines of mice. Future work will include scale-up and distribution of these lines to the scientific community along with the inclusion of other important mouse ES lines in this program.